DNA Replication Initiation and Role of the CDC7/DBF4 Kinase.
In eukaryotes, DNA replication is strictly regulated during cell cycle, occurring once and only once during S phase (Stillman, Science 274, 1659-1664 (1996)). A number of factors conserved in wide varieties of eukaryotes are known to play crucial roles in initiation and elongation stages of DNA replication. These include ORC (Origin Recognition Complex), MCM (Minichromosome Maintenance), Cdc6, Cdc45, RPA (Single-stranded DNA binding proteins), and DNA polymerases. Although assembly of a prereplicative complex (preRC) at an origin, which involves actions of ORC, Cdc6 and MCM proteins, is prerequisite for initiation of DNA replication (Diffley et al., Cell 78, 303-316. (1994); Newlon, Cell 91, 717-720. (1997)), such assembyly is not by itself sufficient for triggering DNA synthesis. Genetic evidence from budding yeast Saccharomyces cerevisiae has indicated that initiation of DNA replication requires action of the serine/threonine CDC7-DBF4 kinase. Molecular studies have now demonstrated that the CDC7/DBF4 kinase has a direct role in DNA replication origin firing.
This evidence is summarised below:
    1) Temperature sensitive cdc7 and dbf4 mutant strains arrest with G1 DNA content after shift to restrictive temperature (Bousset, (1998), [published erratum appears in Genes Dev 1998 Apr 1;12(7):1072]. Genes Dev 12, 480-490).    2) CDC7 activity is required throughout S phase for origin firing; it is therefore not just a molecular switch between G1 and S. i.e. it is required for firing of early and late origins (Bousset et al., supra; Donaldson et al., (1998), Genes Dev 12, 491-501).    3) The DBF4 protein is recruited to origin DNA sequences in vivo (Dowell et al., (1994), [see comments], Science 265, 1243-1246).    4) CDC7/DBF4 kinase preferentially phoshorylates MCM proteins, a component of pre-replicative complexes bound at origins (Sato et al., (1997), EMBO J 16, 4340-4351; Lei et al., (1997), Genes Dev 11, 3365-3374.).    5) This phosphorylation is very likely to be important since a very specific mutation in the MCM5 gene was found to bypass the CDC7/DBF4 kinase requirement (Hardy et al, (1997), Proc Natl Acad Sci USA 94, 3151-3155).
Recently the presence of kinase complexes related to budding yeast Cdc7-Dbf4 has been demonstrated in other eukaryotic organisms including fission yeast, Xenopus and mammals (Masai et al., EMBO J 14, 3094-3104 (1995); Sato et al., EMBO J 16, 4340-4351 (1997); Kim et al., J Biol Chem 273, 23248-23257. (1998); Jiang et al., Proc. Natl. Acad. Sci. USA 94, 14320-14325. (1997); Hess et al., (1998); Gene 211, 133-140; Kumagai et al., Mol. Cell. Biol. 19, 5083-5095. (1999)).
Role of human CDC7/DBF4 complex in DNA replication was assessed using antibody microinjection approach. First, microinjection of specific anti-CDC7 antibody inhibits DNA synthesis in human tumor cells (Jiang et al., EMBO J 18, 5703-5713. (1999)) Secondly, two independent anti-DBF4 antibodies inhibited DNA synthesis when injected into human primary fibroblast KD cells (Kumagai et al., Mol. Cell. Biol. 19, 5083-5095. (1999)). The DNA replication was restored when the antigen was coinjected with antibody. Taken together, mammalian CDC7-related kinase complexes play pivotal roles in cell cycle progression, most likely in S phase initiation, as was discovered in yeast.
Human CDC7/DBF4 Genes and Proteins.
The human full length cDNA encoding for CDC7 kinase homologue was cloned by three different groups:    1) Sato et al. from Tokyo University, Japan, EMBO J 16, 4340-4351. (1997)).    2) Jiang et al. from Salk Institute USA, Proc. Natl. Acad. Sci. USA 94, 14320-14325. (1997).    3) Hess et al. from Pharmacia & Upjohn, Kalamazoo, USA, (1998), Gene 211, 133-140.
The cDNA encoding the human DBF4 subunit was first published in July 1999 by Kumagai et al., Mol. Cell. Biol. 19, 5083-5095. (1999), and more recently by Jiang et al., EMBO J. 18, 5703-5713. (1999).
The CDC7/DBF4 kinase, like cyclin-dependent kinases, is composed by a catalytic subunit CDC7 and a regulatory subunit DBF4, the latter also called ASK1 (activator of S phase kinase) (Kumagai et al., Mol. Cell. Biol. 19, 5083-5095. (1999)). Kinase activity is dependent upon physical interaction between the two subunits, the CDC7 protein alone does not show kinase activity (Kumagai et al., Mol. Cell. Biol. 19, 5083-5095. (1999); Jiang et al., EMBO J 18, 5703-5713. (1999)).
Kinase activity fluctuates during the cell cycle. Since CDC7 levels are constant, this fluctuation might reflect the available amount of activator protein (Kumagai et al., Mol. Cell. Biol. 19, 5083-5095. (1999); Jiang et al., EMBO J 18, 5703-5713. (1999)). No other mechanisms that could regulate the activity of the enzyme have been observed to date but cannot be excluded.
CDC7 is a 574-aminoacid polypeptide that shares a strong homology with S. cerevisiae CDC7 protein (Sato et al., EMBO J 16, 4340-4351. (1997); Jiang et al., Proc. Natl. Acad. Sci. USA 94, 14320-14325. (1997); Hess et al., (1998), Gene 211, 133-140). DBF4 protein has a very limited sequence homology with the yeast counterpart. This homology is confined in two boxes called ASK motive-N (N-terminal) and ASK motif-C (C-terminal). The role of these boxes is not clear although the motif-C is within the interaction domain with CDC7 (Kumagai et al., Mol. Cell. Biol. 19, 5083-5095. (1999)). At present time these two boxes appear to be a unique feature of DBF4 related proteins. Because of the importance of CDC7/DBF4 proteins in DNA replication process it is important to identify genes and proteins with related function or structure and that might regulate activity of CDC7 kinase itself.
The present invention involves the surprising discovery of a novel polypeptide, herein designated DRF1, that exhibits a functional homology to human DBF4/ASK1, and its role as a key component, for example, in regulating CDC7 kinase activity. DRF1 is, thus, useful in the search for novel agents that can modify and/or control DRF1 and/or CDC7 kinase activity. These and other aspects of the invention are described below.